1. Field of the Invention
The present invention relates to methods and apparatus for delivering molecules into a target cell, and, more particularly, to such methods and apparatus for achieving such delivery into cells in vivo through electroporation and electrophoresis.
2. Description of Related Art
The effect of electromagnetic fields on cell membranes has been studied since the 1960s. Early research focused on describing observations that an applied electric field can reversibly break down cell membranes in vitro. Throughout the 1970s the topic was more common in the literature and continued to focus on describing the phenomenon that resulted from brief exposure to intense electric fields as well as the entry of exogenous molecules to the cell interior as a result of membrane breakdown. Applications began to emerge along with a better understanding of reversible membrane breakdown in the 1980s.
Prior research led to the current understanding that exposure of cells to intense electric fields for brief periods of time temporarily destabilized membranes. This effect has been described as a dielectric breakdown due to an induced transmembrane potential, and was termed xe2x80x9celectroporation,xe2x80x9d or xe2x80x9celectropermeabilization,xe2x80x9d because it was observed that molecules that do not normally pass through the membrane gain intracellular access after the cells were treated with electric fields. The porated state was noted to be temporary. Typically, cells remain in a destabilized state on the order of minutes after electrical treatment ceases.
The physical nature of electroporation makes it universally applicable. A variety of procedures utilize this type of treatment, which gives temporary access to the cytosol. These include production on monoclonal antibodies, cell-cell fusion, cell-tissue fusion, insertion of membrane proteins, and genetic transformation. In addition, dyes and fluorescent molecules have been used to investigate the phenomenon of electroporation. A notable example of loading molecules into cells in vivo is electrochemotherapy. The procedure utilizes a drug combined with electric pulses as a means for loading tumor cells with an anticancer drug, and has been performed in a number of animal models and in clinical trials by the present inventors. Also, plasmid DNA has been loaded into rat liver cells in vivo (Heller et al., FEBS Lett. 389, 225-28). The methods published thus far for all in vivo applications utilize multiple direct current pulses that are substantially identical (single amplitude, single pulse duration, and a single duty cycle for delivery). It is known that temporary permeabilization of cell membranes can result if the effects of pulsation fall within two thresholds. First, the intensity of the applied pulses must be above a threshold value in order to electropermeabilize the membranes. This value is cell-type dependent. Second, if the intensity of the treatment is too high, then cells will be killed as a result of membrane damage, which can negate any desired effect of a molecule introduced into the cell. Thus it is critical to apply pulses with intensities that are above the described lower threshold but below the upper threshold in order to impart a temporary permeabilized state.
Protocols for the use of electroporation to load cells in vitro typically use a suspension of single cells or cells that are attached in a planar manner to a growth surface. In vivo electroporation is more complex because tissues are involved. Tissues are composed of individual cells that collectively make up a three-dimensional structure. In either case, the effects on the cell are the same.
FIGS. 1A-1E illustrate details of the electrical treatment procedure. The method comprises:
1. A living biological cell in an electrically conductive medium is positioned between two electrodes (FIG. 1A). The cell""s resting transmembrane potential is indicated by + and xe2x88x92 signs to indicate the separated ionic species that make up the potential.
2. Application of an electrical field (in the form of an applied potential +v) between the two electrodes causes accumulation of charge on either side of the cell. The separated charge adds to the resting potential, resulting in an overall transmembrane potential (resting plus induced). This charge will accumulate as the applied field is increased up to a critical threshold value that is cell-type dependent.
3. If the overall transmembrane potential is increased above this threshold, by applying a field with sufficient magnitude indicated by the pulse +V (FIG. 1C), then the cell membrane is dielectrically broken down. This membrane breakdown has been termed electroporation and/or electropermeabilization. Cells electroporate preferentially in the membrane region that faces the + electrode as the accumulation of charge in this membrane region adds directly to the resting potential. Less poration takes place in the opposite side of the cell because the accumulation of induced charge first cancels the resting potential and then accumulates locally to form an induced potential. Thus a lower total transmembrane potential is induced on this side of the cell, which results in a lower degree of poration.
4. Immediately after electroporation, there is a rapid depolarization of the membrane that takes place as a result of the aqueous electropores (FIG. 1D).
5. Normal membrane fluidity allows the electropores to seal in a time frame that is approximately on the order of minutes (FIG. 1E).
The loading of molecules by electroporation in vitro as well as in vivo is typically carried out by first exposing the cells or tissue of interest to a drug or other molecule (FIG. 2A). The cells or tissue are then exposed to electric fields by administering one or more direct current pulses (FIG. 2B). Electrical treatment is conducted in a manner that results in a temporary membrane destabilization with minimal cytotoxicity. The intensity of electrical treatment is typically described it by the magnitude of the applied electric field. This field is defined as the voltage applied to the electrodes divided by the distance between the electrodes. Electric field strengths ranging from 100 to 5000 V/cm have been successfully used, as reported in the literature, for delivering molecules in vivo and are also specific to the cells or tissue under investigation. Following cessation of stimulation, the pores reseal, with the desired molecules inside the cell (FIG. 2C).
Pulses are usually rectangular in shape; however, exponentially decaying pulses have also been used. The duration of each pulse is called pulse width. Molecule loading has been performed with pulse widths ranging from microseconds (xcexcs) to milliseconds (ms). The number of pulses delivered has ranged from one to eight. Typically, multiple identical pulses of the order of microseconds in duration are utilized during electrical treatment.
For molecules to be delivered to the cell interior by electroporation, it is critical that the molecule of interest be near the exterior of the cell membrane at the time of electroporation. It is also critical to have molecules near all cells within a treated tissue volume in order to provide efficient delivery to all cells within the treatment volume. Currently, molecules are injected intravenously, intraarterially, directly into the treatment site to provide a supply of molecules in the extracellular spaces of the tissues for delivery into the intracellular spaces by electroporation. Other methods for introducing the molecules into the extracellular spaces of tissues such as jet injection and particle bombardment can be used to provide a source of molecules in the extracellular space for delivery using electrical fields.
Currently known delivery methods utilize the electrode systems outlined previously using one or more pulses that are substantially identical with respect to their intensity (V/cm), pulse width, and duty cycle (if more than one pulse). Although protocols that used these types of electrodes and pulses to deliver molecules in vivo have been successful, there are some drawbacks. Pulsing protocols for delivering small molecules such as drugs, in particular, chemotherapeutic agents, have used fields in the range of 1000-5000 V/cm. Such a high-intensity field can cause patient discomfort in the form of pain and/or involuntary muscle movement. Such drug-delivery protocols typically use multiple pulses on the order of microseconds in duration.
There are two types of pulsing protocols that have been used for delivering DNA. The first type is identical to those pulsing protocols used to deliver drugs and suffers from the same patient-related drawbacks. The second type uses pulses on the order of 100-800 V/cm, with pulses lasting up to hundreds of milliseconds. The drawback of these pulses is that their duration can cause great discomfort and also tissue damage.
It is therefore an object of the present invention to provide an improved system and method for delivering molecules to cells in vivo.
It is an additional object to provide such a system and method that do not do not produce the discomfort and tissue damage associated with previously used devices and methods.
These objects and others are achieved by the system and method of the present invention, which combine pulses having different characteristics for delivering molecules to cells in vivo. The pulses of the present invention comprise a high-intensity pulse for inducing electroporation and a low-intensity pulse to induce electrophoretic molecule movement within an interstitial space, molecule adherence to a cell membrane, and electrophoretic movement of the molecule through the permeabilized membrane.
The molecules, which may comprise a unitary charged molecule or a plurality of different charged molecules, may be delivered by a method known in the art, such as by injection combined with electroporation, particle bombardment, and jet injection, although these methods are not intended as limitations. The types of molecules may includes, but are not intended to be limited to, amino acids, bioactive molecules, polypeptides, proteins, antibodies (or fractions thereof), glycoproteins, enzymes, nucleic acids, oligonucleotides, RNA, DNA, competent DNA, plasmid DNA, chromosomes, drugs, other charged organic or inorganic molecules that may or may not have a localized charge region.
The use of a high-intensity and a low-intensity pulse achieves:
1. Equal or better in vivo delivery over conventional pulsing protocols using a single type of pulse
2. Reduction of intensity and/or duration of pulses for inducing electroporation over conventional pulsing protocols using only high-intensity pulses.
3. Decreased muscle stimulation, tissue damage, and patient discomfort.
Target tissue may include, but is not intended to be limited to, normal or abnormal cells within tissues, skin, tumor tissue, or muscle.